Fluoxetine Modulates Apoptosis and ERK1/2 Phosphorylation in Endometrial Cancer In Vitro

Fluoxetine Modulates Apoptosis and ERK1/2 Phosphorylation in Endometrial Cancer In Vitro | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Fluoxetine Modulates Apoptosis and ERK1/2 Phosphorylation in Endometrial Cancer In Vitro Neziha Senem Arı, Ayşe Çakır Gündoğdu, Neslihan Meriç, Orhan Özatik This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8850312/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 8 You are reading this latest preprint version Abstract Background Selective serotonin reuptake inhibitors (SSRIs) are commonly prescribed in oncology settings, yet their direct effects on endometrial cancer cell fate and survival signaling remain unclear. This study examined whether clinically aligned, sub-cytotoxic fluoxetine concentrations modulate viability, cell death phenotype, DNA integrity, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in endometrial cancer cells. Methods Ishikawa endometrial cancer cells and normal human dermal fibroblasts (HDF) were exposed to fluoxetine (100–300 ng/mL) for 48 hours. Metabolic viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death phenotypes were quantified by annexin V and propidium iodide (PI) staining with flow cytometry. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and total ERK1/2 and phosphorylated ERK1/2 (pERK1/2) levels were quantified by immunofluorescence. Results Fluoxetine produced a concentration-dependent reduction in metabolic viability in Ishikawa cells, whereas HDF viability was largely preserved across the same dose range. Flow cytometry demonstrated an increase in PI-positive populations with minimal expansion of the early annexin V–positive fraction in Ishikawa cells, consistent with a late-stage or mixed injury phenotype. TUNEL positivity increased dose-dependently in Ishikawa cells but remained low in HDF cells. Fluoxetine markedly reduced pERK1/2 in Ishikawa cells without a comparable change in total ERK1/2. Conclusion Clinically aligned fluoxetine exposure compromises metabolic fitness and genomic integrity in endometrial cancer cells in parallel with suppression of ERK1/2 activation, while normal fibroblasts show relative resistance under the same conditions. Fluoxetine Endometrial cancer ERK1/2 signaling Phosphorylated ERK1/2 (pERK1/2) Annexin V/propidium iodide TUNEL assay Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. INTRODUCTION Endometrial cancer is among the most frequently diagnosed gynecological malignancies and remains a significant cause of cancer-related morbidity and mortality worldwide. Although early-stage disease is often associated with favorable clinical outcomes, advanced and recurrent forms continue to pose substantial therapeutic challenges [ 1 ]. The biological behavior of endometrial tumors is governed by a complex interplay of hormonal regulation, growth factor signaling, and intracellular stress-response pathways, all of which collectively shape cellular decisions related to proliferation, survival, and death. These signaling networks do not operate in isolation; rather, they dynamically interact to determine whether a cell adapts to stress, enters a quiescent state, or undergoes irreversible death. Understanding how pharmacological agents perturb these networks is therefore critical for identifying novel vulnerabilities in cancer cells and for improving therapeutic strategies [ 2 ]. In parallel with the oncological burden of the disease, psychological distress and depressive symptoms are highly prevalent among patients with cancer. Depression not only compromises quality of life but also negatively influences treatment adherence, patient engagement, and overall clinical outcomes. As a result, antidepressant therapy is frequently prescribed to individuals undergoing cancer treatment [ 3 ]. Among available antidepressants, selective serotonin reuptake inhibitors (SSRIs) are widely used due to their favorable safety profiles and broad clinical efficacy. Fluoxetine, one of the earliest and most extensively prescribed members of this class, has been used for decades in the management of mood disorders. While its primary mechanism of action involves modulation of serotonergic neurotransmission, it is increasingly recognized that fluoxetine also exerts direct biological effects on non-neuronal cells, including immune cells, endothelial cells, and a variety of tumor cell types [ 4 , 5 , 6 ]. This growing recognition has prompted a shift in perspective, whereby antidepressants are no longer viewed solely as neuropsychiatric agents but also as potential modulators of tumor cell biology. Preclinical evidence, including in vitro studies across multiple cancer models (e.g., non–small cell lung cancer, triple-negative breast cancer, bladder cancer, and osteosarcoma) and in vivo validation in some settings, suggests that fluoxetine can influence cell proliferation, oxidative balance, mitochondrial function, and regulated cell death programs [4; 7; 8; 9; 10]. However, the reported effects are not uniform; rather, they appear to be highly context dependent, varying with cell type, concentration, and duration of exposure. In some systems, fluoxetine has been reported to suppress proliferation and induce apoptosis, whereas in others it appears to promote cellular stress responses that may or may not culminate in cell death. This heterogeneity underscores the necessity of tumor-type–specific investigations and highlights the importance of pathway-based studies that move beyond descriptive cytotoxicity assays [ 5 ]. A central signaling axis that warrants particular attention in this context is the mitogen-activated protein kinase/extracellular signal–regulated kinase (MAPK/ERK) pathway. ERK1/2 integrates signals from a broad range of extracellular stimuli, including growth factors, hormones, and stress cues, and translates them into transcriptional programs that regulate proliferation, differentiation, survival, and resistance to therapy [ 11 ]. ERK1/2 is activated through dual phosphorylation of the threonine and tyrosine residues within the conserved Thr–Glu–Tyr (TEY) motif in its activation loop; therefore, pERK1/2 levels are widely used as a practical readout of ERK pathway activation. Activated ERK1/2 can phosphorylate cytosolic substrates and translocate to the nucleus, where it influences transcription factor activity and reshapes gene expression programs linked to proliferation, survival, and stress responses [ 12 ]. Aberrant ERK signaling is a hallmark of many malignancies and has been implicated in tumor progression, metastatic potential, and treatment resistance. [ 13 ]. In endometrial cancer cell models, AF-6/afadin knockdown significantly increased matrigel invasion in Ishikawa (and HEC1A) cells. In Ishikawa cells, this enhanced invasiveness was accompanied by increased phosphorylation of ERK1/2 and Src [ 14 ]. In endometrial cancer, ERK signaling occupies a particularly prominent role. Ishikawa cells, a well-established in vitro model of endometrioid endometrial carcinoma, exhibit strong hormonal responsiveness and active cross-talk between estrogen receptor signaling, phosphoinositide 3-kinase/AKT pathways, and MAPK/ERK cascades. Within this network, ERK1/2 functions not merely as a downstream effector of mitogenic signaling but also as a regulator of cellular survival and apoptotic signaling. Perturbations in ERK phosphorylation can therefore shift the balance between adaptive responses and cell death [ 15 ]. Evaluating how fluoxetine modulates ERK1/2 activation in Ishikawa cells provides a pathway-level entry point into understanding whether this antidepressant reshapes survival signaling in endometrial cancer. A further methodological consideration concerns dose selection. Many in vitro studies of repurposed drugs employ concentrations that greatly exceed clinically achievable levels, which can obscure biologically relevant mechanisms and favor nonspecific cytotoxicity [ 16 ]. In contrast, aligning experimental concentrations with clinically relevant exposure ranges increases the likelihood that observed effects reflect plausible biological interactions rather than generalized toxicity. In addition, the inclusion of a non-malignant comparator cell type enables estimation of a biological safety window and provides insight into whether drug effects preferentially target cancer cells or extend indiscriminately to normal cells. Accordingly, this study was designed to systematically evaluate the in vitro effects of fluoxetine on Ishikawa endometrial cancer cells, focusing on cell viability, cell death phenotype, DNA fragmentation, and ERK1/2 signaling activity within a sub-cytotoxic concentration range. Despite a growing body of literature suggesting an antitumor potential of antidepressants across different cancer models, data addressing the effects of fluoxetine in endometrial cancer cells—particularly in relation to ERK1/2 phosphorylation—remain limited; therefore, the present study aims to fill this knowledge gap [ 6 ]. By including normal human dermal fibroblasts as a non-malignant reference, we sought to interpret the observed responses in terms of selectivity and a potential biological safety window. The integrated assessment of metabolic viability, flow cytometry–based cell death phenotyping, TUNEL, and ERK1/2 phosphorylation/total protein levels is expected to provide a more comprehensive view of how fluoxetine may reshape survival and stress-response signaling in endometrial cancer cells. 2. MATERIALS AND METHODS 2.1. Cell Culture and Maintenance Human endometrial adenocarcinoma Ishikawa cells and normal human dermal fibroblasts (HDF) were used as malignant and non-malignant cellular models, respectively. The cells were obtained from the American Type Culture Collection (ATCC) and maintained under standard culture conditions. Ishikawa cells were cultured in RPMI-1640 medium (Gibco, USA), while HDF cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA). All media were supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and penicillin (100 U/mL) – streptomycin (100 µg/mL) to provide optimal growth conditions and prevent microbial contamination. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂. Routine subculturing was performed using trypsin–EDTA solution once the cells reached approximately 70–80% confluence. For experimental procedures, cells were seeded into 96-well, 24-well, or 6-well culture plates depending on the specific assay requirements. All experiments were conducted using cells within a limited passage range to minimize phenotypic drift. 2.2. Drug Preparation and Dose Selection The fluoxetine was supplied in the form of fluoxetine hydrochloride with ≥ 95% purity (abcr GmbH, Karlsruhe, Germany; product code: AB 401002). Fresh stock solutions were prepared immediately prior to use in the appropriate culture medium for each cell line (RPMI-1640 for Ishikawa cells and DMEM for HDF cells). Dose selection was performed using a stepwise, data-driven approach. In the initial phase, a broad concentration range was determined based on previously published in vitro studies evaluating the biological and antiproliferative effects of fluoxetine in different cancer cell lines; these studies reported that fluoxetine was generally applied at micromolar levels (approximately 5–40 µM) [ 6 , 17 ]. Accordingly, cells were exposed to 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 µM concentrations of fluoxetine hydrochloride for 72 hours, and the dose–response profile was evaluated for preliminary screening. In these preliminary screening experiments, rapid loss of membrane integrity developed in cells, particularly at high micromolar concentrations, and a significant increase in the Annexin V⁻/propidium iodide (PI)⁺ cell population was observed in flow cytometry analyses (data not shown). These findings indicated that cell death at these concentrations predominantly exhibited necrotic characteristics and that early apoptotic responses were limited. Considering that excessive necrotic cell death could mask the evaluation of specific cellular signaling pathways and regulatory biological responses, the experimental approach was reconfigured in the second phase. In this phase, the dose selection was based on the steady-state (Css,ss) plasma concentrations reported for chronic fluoxetine use, and experimental concentrations were created using a 0.1x, 1x, and 10x multiplier approach. The steady-state/plateau concentrations of fluoxetine reported in human plasma after chronic use are approximately 91–302 ng/mL [ 18 ]. In this context, cells were exposed to fluoxetine hydrochloride concentrations of 25, 50, 100, 150, 200, 250, 300, 600, 900, and 1200 ng/mL for 48 hours. Based on the results obtained, doses of 100, 200, and 300 ng/mL, which fall within the reported therapeutic range and allow for the evaluation of cellular responses without causing significant cytotoxicity, were selected for further experiments; it was determined that a dose of 300 ng/mL in this experimental model corresponds to approximately the IC₂₅ level. This approach allowed for the evaluation of sub-cytotoxic biological effects in Ishikawa cells while enabling the preservation of cell viability in healthy HDF cells, thereby establishing a biological and experimental safety window. 2.3. Cell Viability Assay The effects of fluoxetine on cell viability were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with Ishikawa endometrial cancer cells and HDF cells. The MTT assay is a widely used colorimetric and spectrophotometric method for determining cell viability. It is based on the principle that metabolically active cells reduce the yellow MTT to insoluble purple formazan crystals via mitochondrial dehydrogenase enzymes. The amount of formazan formed is considered an indicator of cellular metabolic activity and is directly proportional to the number of viable cells. For MTT analysis, cells were seeded into 96-well plates at a density of 5x10 3 cells per well and allowed to adhere overnight. Each experimental condition was performed in six replicates (n = 6). Cells were treated with fluoxetine. at the concentrations of 100–300 ng/mL. 10% v/v MTT solution was added to each well, and cells were incubated at 37°C for 4 hours. After incubation, the solution was carefully removed, and 100 µL of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formed formazan crystals. The plates were shaken on a heated shaker for 10 minutes to ensure complete dissolution of the crystals [ 19 ]. The absorbance of the resulting formazan dye was measured at 570 nm wavelength using a Beckman Coulter AU680 analyzer (Beckman Coulter, Miami, FL, USA). The optical density (OD) values obtained from the wells treated with fluoxetine were normalized to the control group and calculated as the percentage of live cells. 2.4. Apoptosis Analysis by Flow Cytometry (Annexin V–FITC/PI) Apoptosis and cell death were evaluated by flow cytometry using the Annexin V–FITC/propidium iodide (PI) double staining method. The eBioscience™ Annexin V-FITC Apoptosis Kit (Thermo Fisher Scientific, eBioscience, Cat. No: BMS500FI-100) was used for staining according to the manufacturer's instructions. For analysis, cells were seeded at 5×10⁵ cells/well into 6-well plates and allowed to adhere overnight before being treated with fluoxetine at concentrations of D1 (100 ng/mL), D2 (200 ng/mL), and D3 (300 ng/mL) and incubated for 48 hours. At the end of the treatment, both adherent and suspended cells were collected together, cell pellets were obtained by centrifugation, and the cells were resuspended in 500 µL of 1× binding buffer. For staining, 2 µL Annexin V–FITC and 1 µL PI were added to the cell suspensions; samples were incubated at room temperature in the dark for 5 minutes. After staining, samples were analyzed by flow cytometry, and at least 1×10⁴ events were recorded for each sample. Data were evaluated on dot-plot graphs, and cell populations were classified as live (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and primary necrotic/membrane integrity disrupted (Annexin V⁻/PI⁺); and the results were reported as the percentage distribution of these populations. 2.5. Terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick-end labeling (TUNEL) Analysis Apoptosis analysis was performed using the One-step TUNEL In Situ Apoptosis Kit (Red, Elab Fluor® 594) (Elabscience®, Cat. No: E-CK-A322) according to the manufacturer's instructions. After the relevant procedures, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by permeabilization with 0.1% Triton X-100 for 15 minutes. Subsequently, the cells were incubated with the kit's TUNEL reaction mixture in a humid chamber at 37°C for 30 minutes. The cells were washed with PBS, and a mounting medium containing DAPI (Sigma-Aldrich, #F6057) was used for nuclear counterstaining and sealing the preparations. As a negative control, the TdT enzyme was omitted from the reaction mixture according to the kit protocol, and the procedure was performed under identical conditions. Fluorescent images were acquired at 20× magnification using constant microscope settings and exposure times for all groups. For quantitative evaluation, 5 randomly selected fields from each slide were examined under blind conditions. Cell counts were performed using ImageJ/Fiji v1.52 software (NIH, USA). The apoptotic index was calculated as the percentage of TUNEL-positive cells relative to the total number of DAPI-positive nuclei: Apoptotic Index (AI, %) = (TUNEL⁺ cell count / total DAPI⁺ nucleus count) × 100. 2.6. ERK1/2 and Phospho ERK1/2 Immunofluorescence Analysis Ishikawa and HDF cells were seeded onto 13 mm sterile glass coverslips placed in each well of 24-well culture plates and incubated overnight to ensure adequate cellular adhesion. The next day, the cells were treated at the specified experimental doses for 48 hours. After treatment, the cells were washed with phosphate-buffered saline (PBS; Gibco, #10010-023, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich, #P6148) at room temperature for 20 minutes. Cell membrane permeability was then achieved by treatment with 0.1% Triton X-100 (Sigma-Aldrich, #T8787) for 15 minutes; blocking was performed with 3% bovine serum albumin (BSA) (Sigma-Aldrich, #A9647) for 30 minutes to reduce nonspecific binding. Primary antibody incubation was performed overnight at 4°C using rabbit polyclonal anti-ERK1/2 (Affinity Biosciences, Cat. No: AF0155) and rabbit polyclonal anti-phospho-ERK1/2 (pERK1/2; Thr202/Tyr204) (Affinity Biosciences, Cat. No: AF1015) at a 1:200 dilution for both antibodies. Total ERK1/2 and pERK1/2 staining were performed on separate preparations under the same conditions to prevent potential signal overlap. The following day, after sufficient washing with PBS, the cells were incubated at room temperature for 1 hour with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, #111-585-003) as the secondary antibody. A mounting medium containing DAPI (Sigma-Aldrich, #F6057) was used for nuclear counterstaining. For the negative control, the same staining procedure was performed using only the secondary antibody, without applying the primary antibodies. Imaging was performed using a Zeiss Axio Calibri 7 fluorescence microscope (Carl Zeiss, Germany). All groups were imaged using the same microscope settings and exposure times. Quantitative analysis of fluorescence intensity was performed using ImageJ/Fiji v1.52 software (NIH, USA), and corrected total cell fluorescence (CTCF) was calculated for at least 50 cells per concentration. 2.7. Statistical Analysis GraphPad Prism version 8.4.2 was employed to conduct the statistical analysis. Normality was assessed using the Shapiro–Wilk test, and the data were found not to follow a normal distribution. Therefore, comparisons among groups were performed using the Kruskal–Wallis test, followed by Dunn’s multiple-comparisons test. All tests were two-tailed. P-values less than 0.05 were regarded as statistically significant. Data are presented as mean ± SD. 3. RESULTS 3.1. Dose-dependent effects of fluoxetine on Ishikawa and HDF cell viability In Ishikawa cells, fluoxetine reduced cell viability in a concentration-dependent manner (Fig. 1 A). Viability was determined to be 94% and 91% at 100 ng/mL and 200 ng/mL doses, respectively. The cell viability at 200 ng/mL was significantly lower than that at 100 ng/mL ( p = 0.0232). In the cells treated with 300 ng/mL fluoxetine, a marked decrease in cell viability was detected compared to the untreated ( p < 0.0001) and the 100 ng/mL fluoxetine treated cells ( p = 0.0232). Furthermore, the 300 ng/mL dose was estimated to correspond to approximately the IC₂₅ level. HDF cells serving as the non-malignant control exhibited largely preserved viability after fluoxetine exposure. (Fig. 1 B). Viability at the all three doses remained at levels similar to the control cells, and no significant difference was found in the statistical comparison ( p = 0.0752). These findings indicate that fluoxetine has a cell viability-reducing effect in Ishikawa cells within the selected dose range, while cell viability is largely preserved in HDF cells. 3.2. Annexin V–FITC/PI–Defined Cell Death Patterns and Microscopic Morphology Findings Following fluoxetine administration, the live cell fraction (Annexin V⁻/PI⁻) was found to be highly preserved (approximately 91.5%–96.2%) in the Ishikawa cells, and no statistically significant differences in survival rates was detected between untreated and fluoxetine treated cells. In contrast, a marked increase was observed in the PI + cell fraction with fluoxetine administration. This increase was predominantly in cells located in the Annexin V⁻/PI⁺ region and ranged from approximately 2.6% to 7.6%. Early apoptosis rates (Annexin V⁺/PI⁻) remained low (approximately 0.06%–0.12%). Late apoptotic cells (Annexin V⁺/PI⁺) were detected at limited rates (approximately 0.76%–1.98%). This distribution suggests that the predominant effect of fluoxetine in Ishikawa cells may be related to cell death associated with loss of membrane integrity rather than early apoptosis induction (Fig. 2 B, 2 C). In HDF cells, Following fluoxetine administration in HDF cells, the live cell population (Annexin V⁻/PI⁻) was found to be highly preserved (approximately 99.27–99.67%) and no statistically significant difference was detected compared to the control group. Early apoptotic cells (Annexin V⁺/PI⁻) were observed at low rates (approximately 0.26–0.62%). The necrotic cell fraction (Annexin V⁻/PI⁺) remained at very limited levels (approximately 0.05–0.11%). Late apoptotic cells (Annexin V⁺/PI⁺) were detected at negligible rates. These findings indicate that fluoxetine did not induce significant apoptosis or cell death in HDF cells at the tested doses and durations, and that cell viability was generally preserved (Fig. 2 E, 2 F). Parallel to the flow cytometry findings, when cellular morphological changes associated with fluoxetine treatment were evaluated microscopically; untreated Ishikawa cells exhibited typical epithelial-like morphology, distinct cell-cell junctions, and high confluence. In groups treated with fluoxetine, a decrease in confluence, rounding and shrinking of cells, reduced surface attachment, and increased cellular debris were observed (Fig. 2 A). In contrast, after fluoxetine application in HDF cells, the cells largely retained their typical spindle-shaped fibroblastic morphology, and cell adhesion and overall cellular integrity were not significantly impaired (Fig. 2 D). When these morphological observations were evaluated together with cell population distributions obtained by flow cytometry, they indicated that treatment-related changes were more pronounced in Ishikawa cells. 3.3. Fluoxetine-Associated DNA Fragmentation Following fluoxetine administration to Ishikawa cells, TUNEL positivity (AI, %) elevated with increasing concentrations (Fig. 3 A, 3 B). The percentage of the TUNEL-positive nuclei was increased with the 200 ng/mL fluoxetine, and this increase was more pronounced in the 300 ng/mL concentration (Fig. 3 A). Quantitative analysis showed that the AI in the 200 ng/mL dose increased significantly compared to the untreated control cells ( p = 0.0133). This rise was higher in the 300 ng/mL concentration ( p < 0.0001). Furthermore, the elevation in the 300 ng/mL dose was also significant compared to the 200 ng/mL ( p < 0.0001) (Fig. 3 B). Fluoxetine treatment did not cause a significant change in the AI in HDF cells at any tested dose (Fig. 3 C, 3 D). 3.4. Fluoxetine-Induced Changes in ERK1/2 Phosphorylation ERK1/2 CTCF values in Ishikawa and HDF cells were observed at similar levels between the control and fluoxetine-treated groups ( p = 0.7374, p = 0.8915, Fig. 4 ). In contrast, pERK1/2 CTCF levels decreased significantly with fluoxetine treatment, particularly at high doses. The pERK1/2 CTCF value in the D3 group was significantly lower compared to the control group (p < 0.0001), the D1 group (p < 0.0001), and the D2 group (p = 0.0055) (Fig. 5 A, 5 B). These findings indicate that fluoxetine treatment reduced ERK1/2 phosphorylation in Ishikawa cells while total ERK1/2 levels were preserved. In HDF cells, a decrease in pERK1/2 CTCF levels was observed, particularly in the D3 group. Compared to the control group, a decrease in pERK1/2 was clearly seen in the D3 group ( p = 0.0031) and D2 group (p = 0.0093) (Fig. 5 C, 5 D). These results indicate that high-dose fluoxetine treatment reduces pERK1/2 levels while maintaining total ERK1/2 levels in HDF cells. DISCUSSION The present study investigated the effects of fluoxetine on cellular viability, death phenotype, DNA integrity, and MAPK/ERK signaling in Ishikawa endometrial cancer cells using clinically aligned, sub-cytotoxic concentrations, with human dermal fibroblasts (HDF) as a non-malignant comparator. The principal contribution of this work is the demonstration that fluoxetine attenuates ERK1/2 phosphorylation without altering total ERK1/2 levels and that this signaling modulation coincides with reduced metabolic fitness and increased DNA fragmentation in endometrial cancer cells. Importantly, these effects occur in the absence of a robust early apoptotic signature, suggesting that fluoxetine reshapes survival–stress signaling and cell fate decisions rather than inducing classical apoptosis. A major methodological strength of this study is the dose selection strategy. Many in vitro studies investigating repurposed psychotropic drugs employ supraphysiological micromolar concentrations that primarily induce nonspecific cytotoxicity and membrane rupture, thereby obscuring regulated signaling events [ 20 ]. In contrast, the two-stage dose optimization applied here—beginning with a broad screening and subsequently narrowing the experimental window to ng/mL concentrations aligned with reported steady-state/plateau human plasma concentrations after chronic dosing—allowed the evaluation of biologically relevant stress responses rather than catastrophic cell death [ 21 ]. Under these conditions, Ishikawa cells exhibited a gradual, concentration-dependent decline in metabolic viability, while HDF cells largely preserved viability. Although MTT primarily reflects mitochondrial metabolic activity rather than direct cell number, the parallel reduction in MTT signal together with increased PI positivity and elevated TUNEL staining supports a progressive injury phenotype in Ishikawa cells at these clinically aligned concentrations [ 22 , 23 ]. Analysis of cell death phenotypes by Annexin V–FITC/PI staining revealed that fluoxetine-treated Ishikawa cells did not display a marked increase in the early apoptotic (Annexin V⁺/PI⁻) fraction. Instead, a concentration-dependent increase in PI-positive populations was observed, predominantly within the Annexin V⁻/PI⁺ quadrant. When interpreted in isolation, this pattern could suggest primary necrosis; however, when considered alongside the parallel increase in TUNEL positivity and the reduction in metabolic viability, it is less consistent with acute, nonspecific membrane lysis. Rather, the overall profile supports a late-stage or non-canonical injury/death trajectory in which cells with genomic damage and metabolic compromise ultimately become membrane-permeable [ 23 , 24 ]. At the 48-hour time point, many cells may have already progressed beyond early apoptotic stages, resulting in underrepresentation of Annexin V⁺/PI⁻ populations. Thus, the low early apoptotic fraction should not be interpreted as an absence of apoptosis, but rather as an indication that classical early apoptosis is not the dominant or temporally detectable phenotype under these conditions [ 25 ]. TUNEL analysis provided complementary evidence that fluoxetine exposure is associated with increased DNA strand breaks in Ishikawa cells, whereas HDF cells remained largely unaffected. While TUNEL is widely used to detect DNA fragmentation, TUNEL positivity is not specific to apoptosis and can also label necrotic cell death [ 26 ]. In addition, the TUNEL assay has limited accuracy in differentiating necrotic cells from late-apoptotic cells [ 27 ]. Therefore, its dose-dependent increase, together with declining metabolic viability and altered Annexin V/PI profiles, indicates that fluoxetine compromises genomic integrity in a subset of cancer cells. The discrepancy between prominent DNA fragmentation and limited early apoptotic signaling further supports the notion that fluoxetine induces a mixed or late-stage death phenotype rather than a canonical apoptotic program. Such heterogeneity is increasingly recognized in cancer cell populations, where cells may engage intertwined/overlapping regulated death programs rather than a single uniform pathway [ 23 ], and convergent multi-pathway death phenotypes (e.g., PANoptosis) have been emphasized as a framework for overlapping death features [ 28 ]. The most biologically informative finding of this study is the selective reduction in phosphorylated ERK1/2 levels in fluoxetine-treated Ishikawa cells, while total ERK1/2 abundance remains unchanged. Because ERK1/2 phosphorylation is the canonical activation readout of the MAPK/ERK cascade, this pattern supports attenuation of pathway activity (survival/proliferative signaling) rather than generalized protein loss or staining artifact. [ 29 , 11 ]. ERK signaling is a key node in endometrial cancer biology, integrating growth factor/hormone-related inputs and stress-adaptation cues that collectively support proliferation and treatment resistance [ 30 ]. Consistent with this, ERK1/2 activation has been directly linked to pro-proliferative signaling in endometrial carcinoma models under cytokine stimulation [ 31 ]. In this context, even partial suppression of ERK activation may lower the threshold for cellular decompensation under stress and increase vulnerability to DNA damage–associated cell death, in line with evidence that MAPK/ERK pathway inhibition can expose a DNA-damage vulnerability in cancer cells [ 32 ]. The present data do not establish a causal relationship between ERK1/2 dephosphorylation and the observed death phenotypes; however, the parallel decline in pERK1/2, metabolic fitness, and DNA integrity supports a coherent biological model. Fluoxetine may weaken upstream signaling inputs or shift the kinase–phosphatase balance governing ERK activation, thereby impairing the ability of cancer cells to maintain adaptive responses to intrinsic stress [ 33 ]. In hormonally responsive Ishikawa cells, which show estrogen-driven proliferative responses and ERα-associated phenotype features, disruption of ERK activity may have particularly pronounced consequences [ 34 ]. Given that aberrant MAPK/ERK-related signaling is implicated in endometrial cancer progression and resistance biology, attenuation of pathway activity could plausibly lower the threshold for cellular decompensation under stress [ 30 ]. While the precise upstream mechanisms remain to be elucidated, the preservation of total ERK1/2 strongly suggests that modulation of signaling activity, rather than protein expression, underlies the observed effects [ 33 ]. Comparison with HDF cells provides additional insight into the biological context of these findings. Although a decrease in pERK1/2 was also detected in HDF cells at higher fluoxetine concentrations, this signaling change was not accompanied by significant loss of viability, DNA fragmentation, or overt death phenotypes in our assays. This divergence is consistent with the concept that malignant cells often operate under chronic oncogenic and metabolic stress, becoming highly dependent on stress-adaptive and pro-survival signaling circuits [ 35 ]. In contrast, non-malignant cells can better buffer partial pathway perturbations and may be less reliant on any single survival pathway (the non-oncogene addiction framework) [ 36 ]. Given the signaling plasticity of dermal fibroblasts in maintaining tissue homeostasis [ 37 ], partial attenuation of ERK activity may be tolerated without phenotypic collapse, even though ERK signaling broadly supports growth and survival programs [ 33 ]. Thus, the differential outcomes observed here likely reflect context-dependent signaling vulnerability rather than true drug selectivity. Taken together, these findings support a working model in which fluoxetine attenuates ERK-dependent survival signaling in endometrial cancer cells, leading to reduced metabolic competence and increased susceptibility to irreversible genomic damage [ 33 ]. Rather than triggering a robust early apoptotic response, fluoxetine appears to promote a stress state that evolves toward late-stage or mixed death phenotypes characterized by DNA fragmentation and loss of membrane integrity [ 24 ]. This model aligns with emerging concepts in cancer cell death biology, emphasizing signaling disruption and loss of adaptive capacity over simple induction of apoptosis [ 38 ]. Several limitations of this study should be acknowledged. ERK1/2 and pERK1/2 levels were assessed by immunofluorescence and were not independently validated by immunoblotting. Causal involvement of ERK signaling in the observed phenotypes was not directly tested using pharmacological or genetic modulation. In addition, the findings are restricted to a single endometrial cancer cell line and one non-malignant cell type; extension to additional endometrial cancer models and normal endometrial cells will be necessary to assess generalizability. Despite these limitations, the use of clinically relevant concentrations and integrated phenotypic and signaling analyses provides a solid foundation for future mechanistic studies. In conclusion, this study demonstrates that fluoxetine compromises survival signaling and genomic integrity in Ishikawa endometrial cancer cells through attenuation of ERK1/2 phosphorylation, without inducing classical early apoptosis. These results highlight MAPK/ERK modulation as a potential mechanistic link between SSRI exposure and altered cancer cell fate and underscore the importance of pathway-level analyses when evaluating the non-psychiatric effects of antidepressant drugs in oncologic contexts. Declarations Acknowledgments: None Funding: This study was funded by Kütahya Health Sciences University Scientific Research Projects Unit (Project No: TSA-2023-149). Financial interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions NSA: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Visualization; Writing – original draft. ACG: Methodology; Investigation; Data curation; Writing – review & editing. NM: Methodology; Formal analysis; Writing – review & editing. OÖ: Supervision; Project administration; Resources; Writing – review & editing. All authors reviewed and approved the final version of the manuscript and agree to be accountable for all aspects of the work. References Crosbie EJ, Kitson SJ, McAlpine JN, Mukhopadhyay A, Powell ME, Singh N. Endometrial cancer. Lancet. 2022;399(10333):1412–28. 10.1016/S0140-6736(22)00323-3 . Urick ME, Bell DW. Clinical actionability of molecular targets in endometrial cancer. Nat Rev Cancer. 2019;19(9):510–21. 10.1038/s41568-019-0177-x . Mitchell AJ, Chan M, Bhatti H, Halton M, Grassi L, Johansen C, et al. Prevalence of depression, anxiety, and adjustment disorder in oncological, haematological, and palliative-care settings: a meta-analysis of 94 interview-based studies. Lancet Oncol. 2011;12(2):160–74. 10.1016/S1470-2045(11)70002-X . Radin DP, Patel P. A current perspective on the oncopreventive and oncolytic properties of selective serotonin reuptake inhibitors. Biomed Pharmacother. 2017;87:636–9. 10.1016/j.biopha.2017.01.024 . Bielecka AM, Obuchowicz E. 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Center for the Evaluation of Risks to Human Reproduction (CERHR). NTP-CERHR Monograph on the Potential Human Reproductive and Developmental Effects of Fluoxetine. U.S. Department of Health and Human Services; National Institute of Environmental Health Sciences (NIEHS). November 2004. NIH Publication No. 05-4471. Riss TL, Moravec RA, Niles AL, Duellman S, Benink HA, Worzella TJ, Minor L. (2016). Cell viability assays. Assay Guidance Manual [Internet] . https://www.ncbi.nlm.nih.gov/books/NBK144065/ Robles-Banuelos B, Romo-Perez A, Dominguez-Gomez G, Chavez-Blanco A, Gonzalez-Fierro A, Duenas-Gonzalez A. Selection of clinically relevant drug concentrations for in vitro studies of candidates drugs for cancer repurposing: a proposal. Clin Transl Onco. 2024;26(5):1077–88. 10.1007/s12094-023-03352-w . Sagahón-Azúa J, Medellín-Garibay SE, Chávez-Castillo CE, González-Salinas CG, Milán-Segovia RC, Romano-Moreno S. Factors associated with fluoxetine and norfluoxetine plasma concentrations and clinical response in Mexican patients with mental disorders. Pharmacol Res Perspect. 2021;9(5):e00864. 10.1002/prp2.864 . Ghasemi M, Turnbull T, Sebastian S, Kempson I. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. Int J Mol Sci. 2021;22(23):12827. 10.3390/ijms222312827 . Kari S, Subramanian K, Altomonte IA, Murugesan A, Yli-Harja O, Kandhavelu M. Programmed cell death detection methods: a systematic review and a categorical comparison. Apoptosis. 2022;27(7–8):482–508. 10.1007/s10495-022-01735-y . Costigan HJ, Martin SD. Discriminating between apoptosis, necrosis, necroptosis, and autophagy. Curr Protoc. 2023;3:e951. 10.1002/cpz1.951 . Sabirova S, Sharapova G, Budyukova A, et al. Comprehensive analysis of cellular metrics: From proliferation to mitochondrial membrane potential and cell death in a single sample. Cell Death Discov. 2025;11:119. https://doi.org/10.1038/s41420-025-02391-2 . Sherman MS, McMahon-Skates T, Gaston LS, Katzen SW, Majzoub JA, Goessling W. Harmonizing TUNEL with multiplexed iterative immunofluorescence enriches spatial contextualization of cell death. Cell Rep Methods. 2025;5(5):101047. 10.1016/j.crmeth.2025.101047 . Moldovan C, Mocanu A, Vulpoi A, et al. Current trends in luminescence-based assessment of apoptosis. RSC Adv. 2023;13(45):31641–58. 10.1039/D3RA05809C . Gao J, Xiong A, Liu J, Li X, Wang J, Zhang L, … He X. PANoptosis: bridging apoptosis,pyroptosis, and necroptosis in cancer progression and treatment. Cancer Gene Ther , 2024; 31 (7), 970–983. doi: 10.1038/s41417-024-00765-9. Martin-Vega A, Cobb MH. Navigating the ERK1/2 MAPK cascade. Biomolecules. 2023;13(10):1555. 10.3390/biom13101555 . Chen Y, Jiang L, Zhang L, Chi H, Wang Q. Immune microenvironment and molecular mechanisms in endometrial cancer: implications for resistance and innovative treatments. Discov Oncol. 2025;16(1):532. 10.1007/s12672-025-02169-z . Liu S, Liu L, Lu N, et al. Interleukin-22 promotes endometrial carcinoma cell proliferation and cycle progression via ERK1/2 and p38 activation. Mol Cell Biochem. 2025;480(3):813–25. 10.1007/s11010-024-05318-7 . Zerbib J, et al. Human aneuploid cells depend on the RAF/MEK/ERK pathway for overcoming increased DNA damage. Nat Commun. 2024;15(1):7772. 10.1038/s41467-024-52176-x . Fei J, Guo Y. MAPK/ERK Signaling in Tumorigenesis: mechanisms of growth, invasion, and angiogenesis. EXCLI J. 2025;24:854–79. 10.17179/excli2025-8479 . Pavlič R, Novak Pušić M, Lanišnik Rižner T. Evaluation of the effects of estrogens on endometrial cancer cells of different grades. J Steroid Biochem Mol Biol. 2025;251:106762. 10.1016/j.jsbmb.2025.106762 . Iovanna J, Estaras M, Grasso D, Fernández Zapico ME, Neira JL, Santofimia-Castaño P. Oncogenic stress response mechanisms as new therapeutic targets in cancer treatment: A review. Med (Baltim). 2025;104(24):e42857. 10.1097/MD.0000000000042857 . Di Marco T, Vergaro D, Greco A. COPZ1: an example of non-oncogene addiction in human tumors. Front Pharmacol. 2025;16:1636326. 10.3389/fphar.2025.1636326 . Jipu R, Serban IL, Goriuc A, Jipu AG, Luchian I, Amititeloaie C, … Hurjui LL. Targeting dermal fibroblast senescence: from cellular plasticity to anti-aging therapies. Biomedicines , 2025; 13 (8), 1927. doi.org/10.3390/biomedicines13081927. Peng F, Liao M, Qin R, Zhu S, Peng C, Fu L, … Han B. Regulated cell death (RCD) in cancer: key pathways and targeted therapies. Signal Transduct Target Ther , 2022; 7 (1), 286. doi: 10.1038/s41392-022-01110-y. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 27 Apr, 2026 Reviewers agreed at journal 20 Apr, 2026 Reviews received at journal 14 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 14 Apr, 2026 Editor assigned by journal 12 Feb, 2026 Submission checks completed at journal 12 Feb, 2026 First submitted to journal 11 Feb, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8850312","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":626512724,"identity":"fbbb186f-eb3c-437a-9b91-77513232b308","order_by":0,"name":"Neziha Senem Arı","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABE0lEQVRIie3RsUrDQBzH8QsHyXLF9Q9q8gpXAralL3NBSBZTHB1KzBQHo11TBJ/h3B1ODpIlwTVbDYKTgiAUClI8F7UQU0eH+06/5XN3cAjpdP80+WODDYh8Dvx34n4T83eycaCXbSMD66yRx9PIHljnDK1uh8F8VuWATsZe7Fw8tJFRKpjMcumO0oob6ROEVzDxAZWBF5sWbSO0ZkISU3i8DjkiAsJrIAdgJFKR9pfRRRNLso5O+eKFG+8CAnunVGTdQWqMZC/BjNY9jtUtbBcdKRJ3kNJX5FL2eRlyuSegP88mh0OWB25i+u2kKB7fyDJyaFHdNM8icuC+uqtfp+P9Gc5byUbiazHU+ZM6nU6n29IHcqZgP3gCWSoAAAAASUVORK5CYII=","orcid":"","institution":"Kütahya Sağlık Bilimleri Üniversitesi","correspondingAuthor":true,"prefix":"","firstName":"Neziha","middleName":"Senem","lastName":"Arı","suffix":""},{"id":626512726,"identity":"8d77bcb2-caf2-44c4-8971-ea9af0395167","order_by":1,"name":"Ayşe Çakır Gündoğdu","email":"","orcid":"","institution":"Kütahya Sağlık Bilimleri Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Ayşe","middleName":"Çakır","lastName":"Gündoğdu","suffix":""},{"id":626512728,"identity":"0457a92c-7174-403c-9c28-8a3f4b63648f","order_by":2,"name":"Neslihan Meriç","email":"","orcid":"","institution":"Kütahya Sağlık Bilimleri Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Neslihan","middleName":"","lastName":"Meriç","suffix":""},{"id":626512729,"identity":"9c5fba79-c48a-489f-92ae-63f12514eac7","order_by":3,"name":"Orhan Özatik","email":"","orcid":"","institution":"Kütahya Sağlık Bilimleri Üniversitesi","correspondingAuthor":false,"prefix":"","firstName":"Orhan","middleName":"","lastName":"Özatik","suffix":""}],"badges":[],"createdAt":"2026-02-11 10:33:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8850312/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8850312/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107531317,"identity":"a1b2d8ba-9762-42ac-984b-e0936097daad","added_by":"auto","created_at":"2026-04-22 10:27:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":206378,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of fluoxetine on cell viability in Ishikawa (A) and HDF (B) cells.\u003c/strong\u003e Cells were treated with fluoxetine at concentrations 100 ng/mL, 200 ng/mL, and 300 ng/mL for 48 hours; viability is expressed as a percentage relative to the untreated control cells. Columns represent mean ± SD values. Statistical analysis was performed using the Kruskal–Wallis and Dunn multiple comparison tests; \u003cem\u003ep\u003c/em\u003e values are indicated on the graph.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/3b3c95f6467a7a5cd3979051.png"},{"id":107531329,"identity":"d72535d2-f625-4b67-922a-bf78cff0cb73","added_by":"auto","created_at":"2026-04-22 10:27:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1086262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluoxetine-induced cell death phenotypes in Ishikawa and HDF cells assessed by Annexin V–FITC/PI flow cytometry and morphology.\u003c/strong\u003e (A–C) Ishikawa endometrial cancer cells; (D–F) human dermal fibroblasts (HDF). (A, D) Representative phase-contrast images of cells in the control and fluoxetine-treated groups (D1, D2, D3) after 48 h. Scale bar: 50 µm. (B, E) Representative Annexin V–FITC/PI dot plots showing quadrant-based classification of viable (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic/secondary necrotic (Annexin V⁺/PI⁺), and PI-positive/primary necrotic or membrane-compromised (Annexin V⁻/PI⁺) cell populations. (C, F) Quantification of cell population distribution expressed as percentage of total cells (log10 scale). For each sample, ≥1×10⁴ events were acquired. Statistical significance is indicated on the graphs.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/e9e98b252dbcaadcd6533cec.png"},{"id":107531408,"identity":"1f12ad83-7f00-4d23-8361-f35fc2842464","added_by":"auto","created_at":"2026-04-22 10:27:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":345656,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFluoxetine-induced DNA fragmentation assessed by TUNEL.\u003c/strong\u003e (A, C) Representative fluorescence images of untreated and fluoxetine treated cells: nuclei are shown with DAPI (blue), DNA fragmentation with TUNEL (red); merged images are presented. (B, D) Quantitative analysis of apoptotic index (AI, %) values calculated from the TUNEL-positive cell percentage; statistical significance is indicated in the graph\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/bc3617f5e5887ec036b6c9c2.png"},{"id":107531326,"identity":"1d3107c3-6aff-42bd-abd6-3b9171b74bda","added_by":"auto","created_at":"2026-04-22 10:27:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":508666,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTotal ERK1/2 immunofluorescence analysis in Ishikawa and HDF cells following fluoxetine administration. \u003c/strong\u003e(A, C) Representative fluorescence images of control and fluoxetine groups (D1–D3): nuclei are shown with DAPI (blue), total ERK1/2 immunoreactivity (red); merged images are presented. (B, D) Quantitative analysis of ERK1/2 signal intensity expressed as corrected total cell fluorescence (CTCF); at least 50 cells were analyzed per group. Data are shown as mean ± SD.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/dcb232bdc63737221f63b571.png"},{"id":107531313,"identity":"da7b72ed-def8-414a-945a-5c41b3ac0e2c","added_by":"auto","created_at":"2026-04-22 10:27:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":407735,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunofluorescence analysis of phosphorylated ERK1/2 (pERK1/2) in Ishikawa and HDF cells after fluoxetine administration.\u003c/strong\u003e (A, C) Representative fluorescence images of control and fluoxetine groups (D1–D3): nuclei are shown with DAPI (blue), pERK1/2 (Thr202/Tyr204) immunoreactivity (red); merged images are presented. (B, D) Quantitative analysis of pERK1/2 signal intensity expressed as corrected total cell fluorescence (CTCF); at least 50 cells were analyzed in each group. Data are shown as mean ± SD. Statistical significance is indicated in the graph.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/d6d115836034479db161d9d0.png"},{"id":107706101,"identity":"4bd164e5-7163-4d2c-b871-39c4a412d98c","added_by":"auto","created_at":"2026-04-24 09:17:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2823062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8850312/v1/c17db3b2-5af6-41bb-8a89-827ee8f944fe.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eFluoxetine Modulates Apoptosis and ERK1/2 Phosphorylation in Endometrial Cancer In Vitro \u003c/p\u003e","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eEndometrial cancer is among the most frequently diagnosed gynecological malignancies and remains a significant cause of cancer-related morbidity and mortality worldwide. Although early-stage disease is often associated with favorable clinical outcomes, advanced and recurrent forms continue to pose substantial therapeutic challenges [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The biological behavior of endometrial tumors is governed by a complex interplay of hormonal regulation, growth factor signaling, and intracellular stress-response pathways, all of which collectively shape cellular decisions related to proliferation, survival, and death. These signaling networks do not operate in isolation; rather, they dynamically interact to determine whether a cell adapts to stress, enters a quiescent state, or undergoes irreversible death. Understanding how pharmacological agents perturb these networks is therefore critical for identifying novel vulnerabilities in cancer cells and for improving therapeutic strategies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn parallel with the oncological burden of the disease, psychological distress and depressive symptoms are highly prevalent among patients with cancer. Depression not only compromises quality of life but also negatively influences treatment adherence, patient engagement, and overall clinical outcomes. As a result, antidepressant therapy is frequently prescribed to individuals undergoing cancer treatment [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Among available antidepressants, selective serotonin reuptake inhibitors (SSRIs) are widely used due to their favorable safety profiles and broad clinical efficacy. Fluoxetine, one of the earliest and most extensively prescribed members of this class, has been used for decades in the management of mood disorders. While its primary mechanism of action involves modulation of serotonergic neurotransmission, it is increasingly recognized that fluoxetine also exerts direct biological effects on non-neuronal cells, including immune cells, endothelial cells, and a variety of tumor cell types [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis growing recognition has prompted a shift in perspective, whereby antidepressants are no longer viewed solely as neuropsychiatric agents but also as potential modulators of tumor cell biology. Preclinical evidence, including in vitro studies across multiple cancer models (e.g., non\u0026ndash;small cell lung cancer, triple-negative breast cancer, bladder cancer, and osteosarcoma) and in vivo validation in some settings, suggests that fluoxetine can influence cell proliferation, oxidative balance, mitochondrial function, and regulated cell death programs [4; 7; 8; 9; 10]. However, the reported effects are not uniform; rather, they appear to be highly context dependent, varying with cell type, concentration, and duration of exposure. In some systems, fluoxetine has been reported to suppress proliferation and induce apoptosis, whereas in others it appears to promote cellular stress responses that may or may not culminate in cell death. This heterogeneity underscores the necessity of tumor-type\u0026ndash;specific investigations and highlights the importance of pathway-based studies that move beyond descriptive cytotoxicity assays [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA central signaling axis that warrants particular attention in this context is the mitogen-activated protein kinase/extracellular signal\u0026ndash;regulated kinase (MAPK/ERK) pathway. ERK1/2 integrates signals from a broad range of extracellular stimuli, including growth factors, hormones, and stress cues, and translates them into transcriptional programs that regulate proliferation, differentiation, survival, and resistance to therapy [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ERK1/2 is activated through dual phosphorylation of the threonine and tyrosine residues within the conserved Thr\u0026ndash;Glu\u0026ndash;Tyr (TEY) motif in its activation loop; therefore, pERK1/2 levels are widely used as a practical readout of ERK pathway activation. Activated ERK1/2 can phosphorylate cytosolic substrates and translocate to the nucleus, where it influences transcription factor activity and reshapes gene expression programs linked to proliferation, survival, and stress responses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Aberrant ERK signaling is a hallmark of many malignancies and has been implicated in tumor progression, metastatic potential, and treatment resistance. [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In endometrial cancer cell models, AF-6/afadin knockdown significantly increased matrigel invasion in Ishikawa (and HEC1A) cells. In Ishikawa cells, this enhanced invasiveness was accompanied by increased phosphorylation of ERK1/2 and Src [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn endometrial cancer, ERK signaling occupies a particularly prominent role. Ishikawa cells, a well-established in vitro model of endometrioid endometrial carcinoma, exhibit strong hormonal responsiveness and active cross-talk between estrogen receptor signaling, phosphoinositide 3-kinase/AKT pathways, and MAPK/ERK cascades. Within this network, ERK1/2 functions not merely as a downstream effector of mitogenic signaling but also as a regulator of cellular survival and apoptotic signaling. Perturbations in ERK phosphorylation can therefore shift the balance between adaptive responses and cell death [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Evaluating how fluoxetine modulates ERK1/2 activation in Ishikawa cells provides a pathway-level entry point into understanding whether this antidepressant reshapes survival signaling in endometrial cancer.\u003c/p\u003e \u003cp\u003eA further methodological consideration concerns dose selection. Many in vitro studies of repurposed drugs employ concentrations that greatly exceed clinically achievable levels, which can obscure biologically relevant mechanisms and favor nonspecific cytotoxicity [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, aligning experimental concentrations with clinically relevant exposure ranges increases the likelihood that observed effects reflect plausible biological interactions rather than generalized toxicity. In addition, the inclusion of a non-malignant comparator cell type enables estimation of a biological safety window and provides insight into whether drug effects preferentially target cancer cells or extend indiscriminately to normal cells.\u003c/p\u003e \u003cp\u003eAccordingly, this study was designed to systematically evaluate the in vitro effects of fluoxetine on Ishikawa endometrial cancer cells, focusing on cell viability, cell death phenotype, DNA fragmentation, and ERK1/2 signaling activity within a sub-cytotoxic concentration range. Despite a growing body of literature suggesting an antitumor potential of antidepressants across different cancer models, data addressing the effects of fluoxetine in endometrial cancer cells\u0026mdash;particularly in relation to ERK1/2 phosphorylation\u0026mdash;remain limited; therefore, the present study aims to fill this knowledge gap [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. By including normal human dermal fibroblasts as a non-malignant reference, we sought to interpret the observed responses in terms of selectivity and a potential biological safety window. The integrated assessment of metabolic viability, flow cytometry\u0026ndash;based cell death phenotyping, TUNEL, and ERK1/2 phosphorylation/total protein levels is expected to provide a more comprehensive view of how fluoxetine may reshape survival and stress-response signaling in endometrial cancer cells.\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell Culture and Maintenance\u003c/h2\u003e \u003cp\u003eHuman endometrial adenocarcinoma Ishikawa cells and normal human dermal fibroblasts (HDF) were used as malignant and non-malignant cellular models, respectively. The cells were obtained from the American Type Culture Collection (ATCC) and maintained under standard culture conditions. Ishikawa cells were cultured in RPMI-1640 medium (Gibco, USA), while HDF cells were maintained in Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA). All media were supplemented with 10% fetal bovine serum (FBS), 1% L-glutamine, and penicillin (100 U/mL) \u0026ndash; streptomycin (100 \u0026micro;g/mL) to provide optimal growth conditions and prevent microbial contamination.\u003c/p\u003e \u003cp\u003eCells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂. Routine subculturing was performed using trypsin\u0026ndash;EDTA solution once the cells reached approximately 70\u0026ndash;80% confluence. For experimental procedures, cells were seeded into 96-well, 24-well, or 6-well culture plates depending on the specific assay requirements. All experiments were conducted using cells within a limited passage range to minimize phenotypic drift.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Drug Preparation and Dose Selection\u003c/h2\u003e \u003cp\u003eThe fluoxetine was supplied in the form of fluoxetine hydrochloride with \u0026ge;\u0026thinsp;95% purity (abcr GmbH, Karlsruhe, Germany; product code: AB 401002). Fresh stock solutions were prepared immediately prior to use in the appropriate culture medium for each cell line (RPMI-1640 for Ishikawa cells and DMEM for HDF cells).\u003c/p\u003e \u003cp\u003eDose selection was performed using a stepwise, data-driven approach. In the initial phase, a broad concentration range was determined based on previously published \u003cem\u003ein vitro\u003c/em\u003e studies evaluating the biological and antiproliferative effects of fluoxetine in different cancer cell lines; these studies reported that fluoxetine was generally applied at micromolar levels (approximately 5\u0026ndash;40 \u0026micro;M) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Accordingly, cells were exposed to 5, 10, 15, 20, 25, 30, 35, 40, 45, and 50 \u0026micro;M concentrations of fluoxetine hydrochloride for 72 hours, and the dose\u0026ndash;response profile was evaluated for preliminary screening. In these preliminary screening experiments, rapid loss of membrane integrity developed in cells, particularly at high micromolar concentrations, and a significant increase in the Annexin V⁻/propidium iodide (PI)⁺ cell population was observed in flow cytometry analyses (data not shown). These findings indicated that cell death at these concentrations predominantly exhibited necrotic characteristics and that early apoptotic responses were limited. Considering that excessive necrotic cell death could mask the evaluation of specific cellular signaling pathways and regulatory biological responses, the experimental approach was reconfigured in the second phase. In this phase, the dose selection was based on the steady-state (Css,ss) plasma concentrations reported for chronic fluoxetine use, and experimental concentrations were created using a 0.1x, 1x, and 10x multiplier approach. The steady-state/plateau concentrations of fluoxetine reported in human plasma after chronic use are approximately 91\u0026ndash;302 ng/mL [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In this context, cells were exposed to fluoxetine hydrochloride concentrations of 25, 50, 100, 150, 200, 250, 300, 600, 900, and 1200 ng/mL for 48 hours. Based on the results obtained, doses of 100, 200, and 300 ng/mL, which fall within the reported therapeutic range and allow for the evaluation of cellular responses without causing significant cytotoxicity, were selected for further experiments; it was determined that a dose of 300 ng/mL in this experimental model corresponds to approximately the IC₂₅ level. This approach allowed for the evaluation of sub-cytotoxic biological effects in Ishikawa cells while enabling the preservation of cell viability in healthy HDF cells, thereby establishing a biological and experimental safety window.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Cell Viability Assay\u003c/h2\u003e \u003cp\u003eThe effects of fluoxetine on cell viability were evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay with Ishikawa endometrial cancer cells and HDF cells. The MTT assay is a widely used colorimetric and spectrophotometric method for determining cell viability. It is based on the principle that metabolically active cells reduce the yellow MTT to insoluble purple formazan crystals via mitochondrial dehydrogenase enzymes. The amount of formazan formed is considered an indicator of cellular metabolic activity and is directly proportional to the number of viable cells.\u003c/p\u003e \u003cp\u003eFor MTT analysis, cells were seeded into 96-well plates at a density of 5x10\u003csup\u003e3\u003c/sup\u003e cells per well and allowed to adhere overnight. Each experimental condition was performed in six replicates (n\u0026thinsp;=\u0026thinsp;6). Cells were treated with fluoxetine. at the concentrations of 100\u0026ndash;300 ng/mL. 10% v/v MTT solution was added to each well, and cells were incubated at 37\u0026deg;C for 4 hours. After incubation, the solution was carefully removed, and 100 \u0026micro;L of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formed formazan crystals. The plates were shaken on a heated shaker for 10 minutes to ensure complete dissolution of the crystals [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe absorbance of the resulting formazan dye was measured at 570 nm wavelength using a Beckman Coulter AU680 analyzer (Beckman Coulter, Miami, FL, USA). The optical density (OD) values obtained from the wells treated with fluoxetine were normalized to the control group and calculated as the percentage of live cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Apoptosis Analysis by Flow Cytometry (Annexin V\u0026ndash;FITC/PI)\u003c/h2\u003e \u003cp\u003eApoptosis and cell death were evaluated by flow cytometry using the Annexin V\u0026ndash;FITC/propidium iodide (PI) double staining method. The eBioscience\u0026trade; Annexin V-FITC Apoptosis Kit (Thermo Fisher Scientific, eBioscience, Cat. No: BMS500FI-100) was used for staining according to the manufacturer's instructions. For analysis, cells were seeded at 5\u0026times;10⁵ cells/well into 6-well plates and allowed to adhere overnight before being treated with fluoxetine at concentrations of D1 (100 ng/mL), D2 (200 ng/mL), and D3 (300 ng/mL) and incubated for 48 hours. At the end of the treatment, both adherent and suspended cells were collected together, cell pellets were obtained by centrifugation, and the cells were resuspended in 500 \u0026micro;L of 1\u0026times; binding buffer. For staining, 2 \u0026micro;L Annexin V\u0026ndash;FITC and 1 \u0026micro;L PI were added to the cell suspensions; samples were incubated at room temperature in the dark for 5 minutes. After staining, samples were analyzed by flow cytometry, and at least 1\u0026times;10⁴ events were recorded for each sample. Data were evaluated on dot-plot graphs, and cell populations were classified as live (Annexin V⁻/PI⁻), early apoptotic (Annexin V⁺/PI⁻), late apoptotic (Annexin V⁺/PI⁺), and primary necrotic/membrane integrity disrupted (Annexin V⁻/PI⁺); and the results were reported as the percentage distribution of these populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Terminal deoxynucleotidyl transferase (TdT)\u0026ndash;mediated dUTP nick-end labeling (TUNEL) Analysis\u003c/h2\u003e \u003cp\u003eApoptosis analysis was performed using the One-step TUNEL In Situ Apoptosis Kit (Red, Elab Fluor\u0026reg; 594) (Elabscience\u0026reg;, Cat. No: E-CK-A322) according to the manufacturer's instructions. After the relevant procedures, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by permeabilization with 0.1% Triton X-100 for 15 minutes. Subsequently, the cells were incubated with the kit's TUNEL reaction mixture in a humid chamber at 37\u0026deg;C for 30 minutes. The cells were washed with PBS, and a mounting medium containing DAPI (Sigma-Aldrich, #F6057) was used for nuclear counterstaining and sealing the preparations. As a negative control, the TdT enzyme was omitted from the reaction mixture according to the kit protocol, and the procedure was performed under identical conditions. Fluorescent images were acquired at 20\u0026times; magnification using constant microscope settings and exposure times for all groups.\u003c/p\u003e \u003cp\u003eFor quantitative evaluation, 5 randomly selected fields from each slide were examined under blind conditions. Cell counts were performed using ImageJ/Fiji v1.52 software (NIH, USA). The apoptotic index was calculated as the percentage of TUNEL-positive cells relative to the total number of DAPI-positive nuclei: Apoptotic Index (AI, %) = (TUNEL⁺ cell count / total DAPI⁺ nucleus count) \u0026times; 100.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. ERK1/2 and Phospho ERK1/2 Immunofluorescence Analysis\u003c/h2\u003e \u003cp\u003eIshikawa and HDF cells were seeded onto 13 mm sterile glass coverslips placed in each well of 24-well culture plates and incubated overnight to ensure adequate cellular adhesion. The next day, the cells were treated at the specified experimental doses for 48 hours. After treatment, the cells were washed with phosphate-buffered saline (PBS; Gibco, #10010-023, USA) and fixed with 4% paraformaldehyde (Sigma-Aldrich, #P6148) at room temperature for 20 minutes. Cell membrane permeability was then achieved by treatment with 0.1% Triton X-100 (Sigma-Aldrich, #T8787) for 15 minutes; blocking was performed with 3% bovine serum albumin (BSA) (Sigma-Aldrich, #A9647) for 30 minutes to reduce nonspecific binding.\u003c/p\u003e \u003cp\u003ePrimary antibody incubation was performed overnight at 4\u0026deg;C using rabbit polyclonal anti-ERK1/2 (Affinity Biosciences, Cat. No: AF0155) and rabbit polyclonal anti-phospho-ERK1/2 (pERK1/2; Thr202/Tyr204) (Affinity Biosciences, Cat. No: AF1015) at a 1:200 dilution for both antibodies. Total ERK1/2 and pERK1/2 staining were performed on separate preparations under the same conditions to prevent potential signal overlap. The following day, after sufficient washing with PBS, the cells were incubated at room temperature for 1 hour with Alexa Fluor 594-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, #111-585-003) as the secondary antibody. A mounting medium containing DAPI (Sigma-Aldrich, #F6057) was used for nuclear counterstaining. For the negative control, the same staining procedure was performed using only the secondary antibody, without applying the primary antibodies. Imaging was performed using a Zeiss Axio Calibri 7 fluorescence microscope (Carl Zeiss, Germany). All groups were imaged using the same microscope settings and exposure times. Quantitative analysis of fluorescence intensity was performed using ImageJ/Fiji v1.52 software (NIH, USA), and corrected total cell fluorescence (CTCF) was calculated for at least 50 cells per concentration.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Statistical Analysis\u003c/h2\u003e \u003cp\u003eGraphPad Prism version 8.4.2 was employed to conduct the statistical analysis. Normality was assessed using the Shapiro\u0026ndash;Wilk test, and the data were found not to follow a normal distribution. Therefore, comparisons among groups were performed using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s multiple-comparisons test. All tests were two-tailed. P-values less than 0.05 were regarded as statistically significant. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Dose-dependent effects of fluoxetine on Ishikawa and HDF cell viability\u003c/h2\u003e \u003cp\u003eIn Ishikawa cells, fluoxetine reduced cell viability in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Viability was determined to be 94% and 91% at 100 ng/mL and 200 ng/mL doses, respectively. The cell viability at 200 ng/mL was significantly lower than that at 100 ng/mL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0232). In the cells treated with 300 ng/mL fluoxetine, a marked decrease in cell viability was detected compared to the untreated (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and the 100 ng/mL fluoxetine treated cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0232). Furthermore, the 300 ng/mL dose was estimated to correspond to approximately the IC₂₅ level.\u003c/p\u003e \u003cp\u003eHDF cells serving as the non-malignant control exhibited largely preserved viability after fluoxetine exposure. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Viability at the all three doses remained at levels similar to the control cells, and no significant difference was found in the statistical comparison (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0752).\u003c/p\u003e \u003cp\u003eThese findings indicate that fluoxetine has a cell viability-reducing effect in Ishikawa cells within the selected dose range, while cell viability is largely preserved in HDF cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Annexin V\u0026ndash;FITC/PI\u0026ndash;Defined Cell Death Patterns and Microscopic Morphology Findings\u003c/h2\u003e \u003cp\u003eFollowing fluoxetine administration, the live cell fraction (Annexin V⁻/PI⁻) was found to be highly preserved (approximately 91.5%\u0026ndash;96.2%) in the Ishikawa cells, and no statistically significant differences in survival rates was detected between untreated and fluoxetine treated cells. In contrast, a marked increase was observed in the PI\u003csup\u003e+\u003c/sup\u003e cell fraction with fluoxetine administration. This increase was predominantly in cells located in the Annexin V⁻/PI⁺ region and ranged from approximately 2.6% to 7.6%. Early apoptosis rates (Annexin V⁺/PI⁻) remained low (approximately 0.06%\u0026ndash;0.12%). Late apoptotic cells (Annexin V⁺/PI⁺) were detected at limited rates (approximately 0.76%\u0026ndash;1.98%). This distribution suggests that the predominant effect of fluoxetine in Ishikawa cells may be related to cell death associated with loss of membrane integrity rather than early apoptosis induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eIn HDF cells, Following fluoxetine administration in HDF cells, the live cell population (Annexin V⁻/PI⁻) was found to be highly preserved (approximately 99.27\u0026ndash;99.67%) and no statistically significant difference was detected compared to the control group. Early apoptotic cells (Annexin V⁺/PI⁻) were observed at low rates (approximately 0.26\u0026ndash;0.62%). The necrotic cell fraction (Annexin V⁻/PI⁺) remained at very limited levels (approximately 0.05\u0026ndash;0.11%). Late apoptotic cells (Annexin V⁺/PI⁺) were detected at negligible rates. These findings indicate that fluoxetine did not induce significant apoptosis or cell death in HDF cells at the tested doses and durations, and that cell viability was generally preserved (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003eParallel to the flow cytometry findings, when cellular morphological changes associated with fluoxetine treatment were evaluated microscopically; untreated Ishikawa cells exhibited typical epithelial-like morphology, distinct cell-cell junctions, and high confluence. In groups treated with fluoxetine, a decrease in confluence, rounding and shrinking of cells, reduced surface attachment, and increased cellular debris were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In contrast, after fluoxetine application in HDF cells, the cells largely retained their typical spindle-shaped fibroblastic morphology, and cell adhesion and overall cellular integrity were not significantly impaired (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). When these morphological observations were evaluated together with cell population distributions obtained by flow cytometry, they indicated that treatment-related changes were more pronounced in Ishikawa cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Fluoxetine-Associated DNA Fragmentation\u003c/h2\u003e \u003cp\u003eFollowing fluoxetine administration to Ishikawa cells, TUNEL positivity (AI, %) elevated with increasing concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The percentage of the TUNEL-positive nuclei was increased with the 200 ng/mL fluoxetine, and this increase was more pronounced in the 300 ng/mL concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Quantitative analysis showed that the AI in the 200 ng/mL dose increased significantly compared to the untreated control cells (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0133). This rise was higher in the 300 ng/mL concentration (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Furthermore, the elevation in the 300 ng/mL dose was also significant compared to the 200 ng/mL (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eFluoxetine treatment did not cause a significant change in the AI in HDF cells at any tested dose (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Fluoxetine-Induced Changes in ERK1/2 Phosphorylation\u003c/h2\u003e \u003cp\u003eERK1/2 CTCF values in Ishikawa and HDF cells were observed at similar levels between the control and fluoxetine-treated groups (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.7374, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8915, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn contrast, pERK1/2 CTCF levels decreased significantly with fluoxetine treatment, particularly at high doses. The pERK1/2 CTCF value in the D3 group was significantly lower compared to the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), the D1 group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the D2 group (p\u0026thinsp;=\u0026thinsp;0.0055) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These findings indicate that fluoxetine treatment reduced ERK1/2 phosphorylation in Ishikawa cells while total ERK1/2 levels were preserved.\u003c/p\u003e \u003cp\u003eIn HDF cells, a decrease in pERK1/2 CTCF levels was observed, particularly in the D3 group. Compared to the control group, a decrease in pERK1/2 was clearly seen in the D3 group (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0031) and D2 group (p\u0026thinsp;=\u0026thinsp;0.0093) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results indicate that high-dose fluoxetine treatment reduces pERK1/2 levels while maintaining total ERK1/2 levels in HDF cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe present study investigated the effects of fluoxetine on cellular viability, death phenotype, DNA integrity, and MAPK/ERK signaling in Ishikawa endometrial cancer cells using clinically aligned, sub-cytotoxic concentrations, with human dermal fibroblasts (HDF) as a non-malignant comparator. The principal contribution of this work is the demonstration that fluoxetine attenuates ERK1/2 phosphorylation without altering total ERK1/2 levels and that this signaling modulation coincides with reduced metabolic fitness and increased DNA fragmentation in endometrial cancer cells. Importantly, these effects occur in the absence of a robust early apoptotic signature, suggesting that fluoxetine reshapes survival\u0026ndash;stress signaling and cell fate decisions rather than inducing classical apoptosis.\u003c/p\u003e \u003cp\u003eA major methodological strength of this study is the dose selection strategy. Many in vitro studies investigating repurposed psychotropic drugs employ supraphysiological micromolar concentrations that primarily induce nonspecific cytotoxicity and membrane rupture, thereby obscuring regulated signaling events [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast, the two-stage dose optimization applied here\u0026mdash;beginning with a broad screening and subsequently narrowing the experimental window to ng/mL concentrations aligned with reported steady-state/plateau human plasma concentrations after chronic dosing\u0026mdash;allowed the evaluation of biologically relevant stress responses rather than catastrophic cell death [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Under these conditions, Ishikawa cells exhibited a gradual, concentration-dependent decline in metabolic viability, while HDF cells largely preserved viability. Although MTT primarily reflects mitochondrial metabolic activity rather than direct cell number, the parallel reduction in MTT signal together with increased PI positivity and elevated TUNEL staining supports a progressive injury phenotype in Ishikawa cells at these clinically aligned concentrations [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnalysis of cell death phenotypes by Annexin V\u0026ndash;FITC/PI staining revealed that fluoxetine-treated Ishikawa cells did not display a marked increase in the early apoptotic (Annexin V⁺/PI⁻) fraction. Instead, a concentration-dependent increase in PI-positive populations was observed, predominantly within the Annexin V⁻/PI⁺ quadrant. When interpreted in isolation, this pattern could suggest primary necrosis; however, when considered alongside the parallel increase in TUNEL positivity and the reduction in metabolic viability, it is less consistent with acute, nonspecific membrane lysis. Rather, the overall profile supports a late-stage or non-canonical injury/death trajectory in which cells with genomic damage and metabolic compromise ultimately become membrane-permeable [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. At the 48-hour time point, many cells may have already progressed beyond early apoptotic stages, resulting in underrepresentation of Annexin V⁺/PI⁻ populations. Thus, the low early apoptotic fraction should not be interpreted as an absence of apoptosis, but rather as an indication that classical early apoptosis is not the dominant or temporally detectable phenotype under these conditions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTUNEL analysis provided complementary evidence that fluoxetine exposure is associated with increased DNA strand breaks in Ishikawa cells, whereas HDF cells remained largely unaffected. While TUNEL is widely used to detect DNA fragmentation, TUNEL positivity is not specific to apoptosis and can also label necrotic cell death [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, the TUNEL assay has limited accuracy in differentiating necrotic cells from late-apoptotic cells [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Therefore, its dose-dependent increase, together with declining metabolic viability and altered Annexin V/PI profiles, indicates that fluoxetine compromises genomic integrity in a subset of cancer cells. The discrepancy between prominent DNA fragmentation and limited early apoptotic signaling further supports the notion that fluoxetine induces a mixed or late-stage death phenotype rather than a canonical apoptotic program. Such heterogeneity is increasingly recognized in cancer cell populations, where cells may engage intertwined/overlapping regulated death programs rather than a single uniform pathway [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and convergent multi-pathway death phenotypes (e.g., PANoptosis) have been emphasized as a framework for overlapping death features [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe most biologically informative finding of this study is the selective reduction in phosphorylated ERK1/2 levels in fluoxetine-treated Ishikawa cells, while total ERK1/2 abundance remains unchanged. Because ERK1/2 phosphorylation is the canonical activation readout of the MAPK/ERK cascade, this pattern supports attenuation of pathway activity (survival/proliferative signaling) rather than generalized protein loss or staining artifact. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. ERK signaling is a key node in endometrial cancer biology, integrating growth factor/hormone-related inputs and stress-adaptation cues that collectively support proliferation and treatment resistance [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Consistent with this, ERK1/2 activation has been directly linked to pro-proliferative signaling in endometrial carcinoma models under cytokine stimulation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this context, even partial suppression of ERK activation may lower the threshold for cellular decompensation under stress and increase vulnerability to DNA damage\u0026ndash;associated cell death, in line with evidence that MAPK/ERK pathway inhibition can expose a DNA-damage vulnerability in cancer cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present data do not establish a causal relationship between ERK1/2 dephosphorylation and the observed death phenotypes; however, the parallel decline in pERK1/2, metabolic fitness, and DNA integrity supports a coherent biological model. Fluoxetine may weaken upstream signaling inputs or shift the kinase\u0026ndash;phosphatase balance governing ERK activation, thereby impairing the ability of cancer cells to maintain adaptive responses to intrinsic stress [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In hormonally responsive Ishikawa cells, which show estrogen-driven proliferative responses and ERα-associated phenotype features, disruption of ERK activity may have particularly pronounced consequences [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Given that aberrant MAPK/ERK-related signaling is implicated in endometrial cancer progression and resistance biology, attenuation of pathway activity could plausibly lower the threshold for cellular decompensation under stress [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. While the precise upstream mechanisms remain to be elucidated, the preservation of total ERK1/2 strongly suggests that modulation of signaling activity, rather than protein expression, underlies the observed effects [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eComparison with HDF cells provides additional insight into the biological context of these findings. Although a decrease in pERK1/2 was also detected in HDF cells at higher fluoxetine concentrations, this signaling change was not accompanied by significant loss of viability, DNA fragmentation, or overt death phenotypes in our assays. This divergence is consistent with the concept that malignant cells often operate under chronic oncogenic and metabolic stress, becoming highly dependent on stress-adaptive and pro-survival signaling circuits [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast, non-malignant cells can better buffer partial pathway perturbations and may be less reliant on any single survival pathway (the non-oncogene addiction framework) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Given the signaling plasticity of dermal fibroblasts in maintaining tissue homeostasis [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], partial attenuation of ERK activity may be tolerated without phenotypic collapse, even though ERK signaling broadly supports growth and survival programs [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Thus, the differential outcomes observed here likely reflect context-dependent signaling vulnerability rather than true drug selectivity.\u003c/p\u003e \u003cp\u003eTaken together, these findings support a working model in which fluoxetine attenuates ERK-dependent survival signaling in endometrial cancer cells, leading to reduced metabolic competence and increased susceptibility to irreversible genomic damage [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Rather than triggering a robust early apoptotic response, fluoxetine appears to promote a stress state that evolves toward late-stage or mixed death phenotypes characterized by DNA fragmentation and loss of membrane integrity [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This model aligns with emerging concepts in cancer cell death biology, emphasizing signaling disruption and loss of adaptive capacity over simple induction of apoptosis [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral limitations of this study should be acknowledged. ERK1/2 and pERK1/2 levels were assessed by immunofluorescence and were not independently validated by immunoblotting. Causal involvement of ERK signaling in the observed phenotypes was not directly tested using pharmacological or genetic modulation. In addition, the findings are restricted to a single endometrial cancer cell line and one non-malignant cell type; extension to additional endometrial cancer models and normal endometrial cells will be necessary to assess generalizability. Despite these limitations, the use of clinically relevant concentrations and integrated phenotypic and signaling analyses provides a solid foundation for future mechanistic studies.\u003c/p\u003e \u003cp\u003eIn conclusion, this study demonstrates that fluoxetine compromises survival signaling and genomic integrity in Ishikawa endometrial cancer cells through attenuation of ERK1/2 phosphorylation, without inducing classical early apoptosis. These results highlight MAPK/ERK modulation as a potential mechanistic link between SSRI exposure and altered cancer cell fate and underscore the importance of pathway-level analyses when evaluating the non-psychiatric effects of antidepressant drugs in oncologic contexts.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e None\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This study was funded by K\u0026uuml;tahya Health Sciences University Scientific Research Projects Unit (Project No: TSA-2023-149).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eFinancial interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003cbr\u003e\u0026nbsp;NSA: Conceptualization; Methodology; Investigation; Data curation; Formal analysis; Visualization; Writing \u0026ndash; original draft. 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Regulated cell death (RCD) in cancer: key pathways and targeted therapies. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e, 2022;\u003cem\u003e7\u003c/em\u003e(1), 286. doi: 10.1038/s41392-022-01110-y.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Fluoxetine, Endometrial cancer, ERK1/2 signaling, Phosphorylated ERK1/2 (pERK1/2), Annexin V/propidium iodide, TUNEL assay","lastPublishedDoi":"10.21203/rs.3.rs-8850312/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8850312/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eSelective serotonin reuptake inhibitors (SSRIs) are commonly prescribed in oncology settings, yet their direct effects on endometrial cancer cell fate and survival signaling remain unclear. This study examined whether clinically aligned, sub-cytotoxic fluoxetine concentrations modulate viability, cell death phenotype, DNA integrity, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in endometrial cancer cells.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIshikawa endometrial cancer cells and normal human dermal fibroblasts (HDF) were exposed to fluoxetine (100\u0026ndash;300 ng/mL) for 48 hours. Metabolic viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell death phenotypes were quantified by annexin V and propidium iodide (PI) staining with flow cytometry. DNA fragmentation was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL), and total ERK1/2 and phosphorylated ERK1/2 (pERK1/2) levels were quantified by immunofluorescence.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eFluoxetine produced a concentration-dependent reduction in metabolic viability in Ishikawa cells, whereas HDF viability was largely preserved across the same dose range. Flow cytometry demonstrated an increase in PI-positive populations with minimal expansion of the early annexin V\u0026ndash;positive fraction in Ishikawa cells, consistent with a late-stage or mixed injury phenotype. TUNEL positivity increased dose-dependently in Ishikawa cells but remained low in HDF cells. Fluoxetine markedly reduced pERK1/2 in Ishikawa cells without a comparable change in total ERK1/2.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eClinically aligned fluoxetine exposure compromises metabolic fitness and genomic integrity in endometrial cancer cells in parallel with suppression of ERK1/2 activation, while normal fibroblasts show relative resistance under the same conditions.\u003c/p\u003e","manuscriptTitle":"Fluoxetine Modulates Apoptosis and ERK1/2 Phosphorylation in Endometrial Cancer In Vitro","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-22 10:26:38","doi":"10.21203/rs.3.rs-8850312/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-27T11:30:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"35713652848789482859452537253175888452","date":"2026-04-20T17:35:15+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-14T17:23:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"300459141436372434827941224393027217291","date":"2026-04-14T16:19:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-14T14:36:04+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-12T10:26:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-12T10:22:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2026-02-11T09:56:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"d933ac9f-0c98-4d4a-8c61-5803f7974bc3","owner":[],"postedDate":"April 22nd, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T10:26:38+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-22 10:26:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8850312","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8850312","identity":"rs-8850312","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}